Project Summary Multiple sclerosis (MS) is likely caused by a combination of genetic and environmental factors; however, the mechanisms contributing to these factors remain poorly understood. Epstein-Barr virus (EBV) in particular is a well-established environmental risk factor for MS. We have created a computational algorithm that systematically searches for common molecular mechanisms that might be impacted at multiple MS-associated loci. Using this algorithm, we have discovered that over 40% of MS-associated loci contain MS genetic variants that fall within regions of the human genome occupied by the EBV-encoded EBNA2 protein (44 out of 109, >4.6- fold enrichment, P<10-25). Other top proteins include known EBNA2 human interacting proteins (RBPJ, RELA, SPI1) and proteins recently shown to participate in ?EBV super-enhancers?, which enable proliferation and survival of EBV infected B cells. The same MS-associated variants also impact gene expression levels of MS- associated genes in EBV-infected B cell lines. Our hypothesis is that allele-dependent binding of EBNA2 and its co-factors explains the allele-dependent risk at many MS genetic loci. Importantly, this hypothesis links the genetic associations of MS to the known molecular roles played by EBV and Notch signaling. In total, >40 of the known MS associations might be explained by this common mechanism uniting the genetic and the environmental risk components of MS. We propose the following Aims. Aim 1. Genome-wide experimental assessment of allele-dependent EBNA2 and human protein binding to risk alleles at MS loci. We will examine allele-dependent protein binding in MS patient-derived EBV- transformed B cell lines and in T cells using ChIP-seq. We will identify genome-wide allele-dependent co-binding of EBNA2 with its partners using our new, innovative Split Dam ID-seq technique. Resulting data will be used to create an improved model of MS genetic risk mechanism and results will be freely disseminated. Aim 2. Discover specific MS-associated loci where allele-dependent EBNA2 or human protein-DNA interactions result in allele-dependent gene expression. We will assess allele-dependent binding of these proteins at likely causal variants, and allele-dependent gene expression by multiple experimental approaches. Aim 3. Establish causality for intermediate phenotypes at selected MS-associated variants. We will establish the causal effect of these variants by demonstrating the necessity and sufficiency of candidate risk alleles in MS patient-derived cells through a novel dead Cas9 activation system, and by performing genome editing with CRISPR/Cas9 followed by gene expression monitoring. The concept that disease might be influenced by allele-dependent assembly of protein complexes controlled by a virus is highly innovative and has never before been experimentally demonstrated. Results from this proposal would provide strong rationale to develop therapies that interfere with EBNA2 binding, or vaccines that prevent EBV infection, offering the prospect of eliminating MS.